The present invention pertains to the analysis of biomolecular samples. More particularly, the present invention relates to fabrication and integration of thermal management techniques and devices for close proximity monitoring of a bioassay in a bioelectronic analyzer for the analysis of biomolecular samples such as nucleic acids.
Molecular biology is an ever expanding field of study. Of great importance within the field of molecular biology is the detection and analysis of RNA, DNA, bacteria, proteins, and the like. Identification of molecular structure has become very important in many industries. In particular, biological molecules such as nucleic acids and proteins are analyzed to form the basis of clinical diagnostic assays. Currently it is predicted that a large market exits for bio-chips (micro-array chips) in the diagnosing and treating of diseases. Envisioned is a day when physicians will have the capabilities to use bio-chips to make an immediate genomic marker based diagnosis in their offices without the need for a lab as an intermediate diagnostic facility.
Currently, the greatest emphasis and market existence for bio-chips is within the field of genetic and pharmaceutical research, where many thousands of genes can be analyzed in parallel. The procedures utilized often involve large numbers of repetitive steps which consume large amounts of time. With the advent of large projects such as the human genome project, faster and less complex techniques are required.
Simpler and quicker analysis of molecules has been provided by the development of devices often referred to as biochips, which include arrays of test sites formed on a substrate platform. Each of the plurality of test sites includes probes therein to bond with target molecules from samples applied to the device. During analyzation, once certain conditions are met (discussed presently), the binding of a biomolecular to a probe is noted, thereby providing for the identification of the specific biomolecular.
DNA chips or microarrays generally consist of thin wafers of glass, silicon, plastic, printed circuit board (PCB), or ceramic having numerous microscopic bits of bio-molecules or porous support medium containing biomolecules, such as immobilized DNA probes sequences arrayed on the surface. These are used to identify specific disease genes and to speed drug discovery efforts. For example, microarray data has been used to identify gene clusters based on co-expression (Eisen, M. B. et al., Proc. Natl. Acad. Sci., 95, p. 14863-8, (1998)), define metrics that measure a gene""s involvement in a particular cellular event or biochemical process (Spellman, P. T., et al., Molecular Biology of the Cell, 9, p. 3273-97, (1998)) and predict regulatory elements (Brazma, A., et al., Genome Research, 8, p. 1202-15, (1998)). It is anticipated that in the future increased use of bio-molecule related science will allow for a more personalized practice of medicine, more particularly the design and use of customized treatments and therapies based on a patient""s genetic makeup (for review see Health Horizons articles on http://www.msnbc.com/news/horizons_front.html).
Currently, bio-chips, more specifically DNA chips, are known that are based on a common method of manufacture, namely the etching of silicon computer chips, as currently utilized in the semiconductor industry (O""Donnell-Maloney, Maryanne J. et al., Tibtech, 14, p. 401-407, (1996)). Of all of the uses of bio-chips to study bio-molecules, the study of DNA is the most mature. In one specific instance, a photoactivated DNA probe synthesis process is used to manufacture high density DNA chips (Fodor et al., Science, 251, p. 767-773, (1991)). Typically eighty photolithographic mask levels are used to synthesize DNA probes. Alternative approaches for dispensing reagents on a substrate have been reported in the prior art (e.g. U.S. Pat. No. 6,048,699, issued Apr. 11, 2000; U.S. Pat. No. 6,013,446, issued Nov. 1, 2000). In particular, the use of dispensing techniques to place purified, presynthesized oligonucleotides onto specific locations on a surface to produce a DNA chip is described in Schober, A., et al., BioTechniques, 15(2), p. 324-329, (1993) and U.S. Pat. No. 6,083,762, issued Jul. 4, 2000. The later technique does not require photolithography and requires fewer redundant probes because the purity of the probe sequences is much higher than in the photoactivated probe synthesis process. [E.P. 0910570 A1, issued Apr. 28, 1999; U.S. Pat. No. 6,312,960, issued Nov. 6, 2001].
An alternative means for synthesizing DNA probes is by using tiny micromirrors which allow for the placement of in excess of 300,000 bits of DNA onto a chip in just a few hours. In addition, the use of ink-jet printing is known, using high-speed robotic devices to print DNA on tiny squares of glass, to form an array. (U.S. Pat. No. 6,285,490B1, issued on Sep. 4, 2001). These types of machines are capable of forming as many as 32,000 DNA molecules on a single chip.
Still other methods include the use of fiber-optic bundles to build chips capable of holding 50,000 different DNA fragments on a single chip, and microelectronic chips that utilize electricity to attach DNA molecules to the surface of the chip. Other techniques can comprises the use of RF transponders, (U.S. Pat. No. 5,981,166A1, issued on Nov. 9, 1999), microbeads (Czarnik et al., Modern Drug Discovery, 1(2), p. 49-55 (1998)); U.S. Pat. No. 6,266,459B1, issued Jul. 24, 2001; U.S. Pat. No. 6,261,782B1, issued Jul. 17, 2001).
In the typical application, the biomolecular, or biological, sample that is being tested must be heated while held in the biochip to enhance kinetics prior to analyzation. In most instances, an external separate probe for temperature measurement is utilized to monitor the temperature of the biomolecular sample while the sample and biochip are placed in an oven or in conjunction with an external power source generating heating of the entire biochip or part thereof (e.g. the use of a Peltier heater/cooler requires mass transfer through at least the mass of the substrate of the chip on which said sample is attached that can typically limit the efficiency and duration of the thermal process to about 1 C/s). More particularly, the probe (e.g. resistance temperature device (RTD), pn junction, electrode, Kelvin probe, (e.g. used in atomic force microscopy) (AFM)) is in thermal contact with the biomolecular sample which must be heated to a given temperature and held at that temperature for a given period for analyzation of the sample to take place. Typically, a separate, externally located Peltier cooler/heater is utilized to accomplish this heating of the biomolecular sample and maintenance of the sample at the appropriate temperature. Prior to use, the heater must undergo calibration so that proper temperature sensing is achieved. (or alternative differential measurement). The bio-chip, containing the DNA probes and the biomolecular sample is introduced into a pre-calibrated oven chamber or preferably a calibrated Peltier heater, where the temperature is sensed and adjusted so that proper analyzation can take place. This presents not only an additional analyzation step, but a delay in a xe2x80x9csample in, data outxe2x80x9d cycle. Furthermore, there is no control of the thermal profile at the chip level yielding to possible uncontrolled inhomogeneous thermal gradient at the sensor pad of the microarray of the biochip. More recently, Kajiyama et al. (Kajiyama, T., et al, Micro Total Analysis Systems 2000, p. 505-508, (2000) and E.P. No. 1108472A2, published on Jun. 20, 2001) described how to arrange DNA probes based on their melting temperature and hybridization using Si-islands that can be independently controlled by using the simple function of the pn-junction""s voltage. Although, Kajiyama is teaching a method for attaching oligonucleotide probes to a silicon nitride surface, the method requires (i) chemically modifying the silicon nitride for generating reactive amino groups on the surface (ii) depositing probes at concentration greater than 25 xcexcM and (iii) using dye-tagged PCR products for fluorescence scanning detection. Such an approach is costly, slow and not easily integratable. The present invention aims to overcome most of the limitations by combining highly sensitive and labeless bio-electronic detection of nucleic acid (e.g. single-stranded DNA) with high spatial thermal actuation and monitoring of a biochemical reaction. Continuous monitoring of a target binding process and quantitative measurements as well as the high degree of integration of such a platform are some of the critical improvements described in the present invention. The proposed apparatus doesn""t require any washing steps that can interfere with in-situ thermal profiling as it has been published in the prior art.
Optical analysis of the biomolecular sample is typically utilized with conventional DNA chips. Many instances in the prior art require the binding of a fluorescent marker to the sample so that the amount of fluorescence of the marker bound to a diagnostic probe can be measured with an optical microscope system [Corle, G. S., and Kino, G. S., Confocal Scanning Optical Microscopy and Related Imaging Systems, Chapter 1.3, Academic Press 1996]. An optical system, such as a confocal-point microscope or an epifluorescence imaging microscope, may be utilized and the amount of the bonded sample is calculated on the basis of the amount of the fluorescence. Typically, the operation of most commercially available DNA chip scanners comprise an epifluorescence imaging microscope, a substantially planar wavelength-selective mirror holder disposed askew to a common optical pathway, the holder having a plurality of mirrors mounted thereon and having an axle attached normal thereto for rotating the holder so as to place a selected one of the mirrors in the common optical pathway. A motor is connected to the axle for rotating the mirror holder, either selectively or continuously. When a selected mirror is placed in the common optical pathway, the pathway is split so that an excitation light beam of one wavelength traveling along a source optical pathway from a light source is reflected by the mirror along the common optical pathway toward a sample (e.g. DNA spots of a microarray), while fluorescence light produced by the sample and directed back along the common optical pathway passes through the mirror to travel along a detector optical pathway to a detector (e.g. CCD camera). [Basarsky, T., Verdnik, D., Zhai, J. Y., and Wellis, D., Microarray Biochip Technology, p. 265-284, BioTechniques Books, edited by Mark Schena, Eaton Publishing (2000)]. Most of the fluorescence-based bioassay also require typically several labeling steps to incorporate some organic dye molecules within preferably a DNA sample. These steps are often costly, labor intensive and they can result in errors (e.g. loss of specificity due to unspecific binding).
It would be highly advantageous, therefore, to remedy the foregoing and other deficiencies inherent in the prior art. Accordingly, it is an object of the present invention to provide a new and improved apparatus for analyzing a biomolecular sample with integrated controlled thermal conditions, including a plurality of bio-molecules, and method for fabrication and use thereof.
Another object of the present invention is to provide a method and apparatus for analyzing bio-molecules in which an integrated biochip, including at least one integrated thermal sensor, is utilized to achieve on-chip direct temperature measurements and biosensing.
And another object of the present invention is to provide a method and apparatus for analyzing bio-molecules which is fast and efficient by controlling the environmental conditions (e.g. temperature, moisture, fluid mixing properties) of a sample in near proximity of a sensor array.
A further object of the present invention is to provide a method and apparatus for analyzing bio-molecules wherein the analyzing of the bio-molecules includes an integrated bio chip, including at least one thermal sensor, which is utilized to achieve on-chip direct temperature measurements and biosensing.
A further object of the present invention is to provide for a method and apparatus wherein biosensing is performed preferably by electronic detection of biological reactions such as nucleic acid hybridization (e.g. DNA, RNA) or protein interactions (e.g. immunoassay).
A still further object of the present invention is to provide for a method and apparatus for analyzing bio-molecules that provides for rapid deployment of an accurate analysis device with minimal manufacturing difficulty.